Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis

An O₂ electrode system with a specially designed chamber for `whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O₂ concentrations on photosynthetic O₂ evolution. At pH = 5.5 and 20% O₂, cells grown in HCO₃⁻ medium (low CO₂, pH ≥ 9.0) exhibited a higher affinity for external CO₂ (K_½(CO₂) = 40 ± 6 micromolar) than the cells grown for at least 24 hours in high-CO₂ medium (pH = 6.5), (K_½(CO₂) = 94 ± 16 micromolar). With O₂ ≤ 2% in contrast, both types of cells showed a high apparent affinity (K_½(CO₂) = 50 − 52 micromolar). A Warburg effect was detectable only in the low affinity cells previously cultivated in high-CO₂ medium (pH = 6.5). The high-pH, HCO₃⁻-grown cells, when exposed to low pH (5.5) conditions, exhibited a response indicating an ability to fix CO₂ which exceeded the CO₂ externally supplied, and the reverse situation has been observed in high-CO₂-grown cells. At pH 8.2, the apparent photosynthetic affinity for external HCO₃⁻ (K_½[HCO₃⁻]) was 0.6 ± 0.2 millimolar, at 20% O₂. But under low O₂ concentrations (≤2%), surprisingly, an inhibition of net O₂ evolution was elicited, which was maximal at low HCO₃⁻ concentrations. These results indicate that: (a) photorespiration occurs in this alga and can be revealed by cultivation in high-CO₂ medium, (b) Chara cells are able to accumulate CO₂ internally by means of a process apparently independent of the plasmalemma HCO₃⁻ transport system, (c) molecular oxygen appears to be required for photosynthetic utilization of exogenous HCO₃⁻: pseudocyclic electron flow, sustained by O₂ photoreduction, may produce the additional ATP needed for the HCO₃⁻ transport.     

Effect of ATPase inhibitors on cell potential and k influx in corn roots

Experiments were performed to determine the effect of plasmalemma ATPase inhibitors on cell potentials (Ψ) and K⁺ (⁸⁶Rb) influx of corn root tissue over a wide range of K⁺ activity. N,N′Dicyclohexylcarbodiimide (DCCD), oligomycin, and diethylstilbestrol (DES) pretreatment greatly reduced active K⁺ influx and depolarized Ψ at low, but not at high, K⁺ activity (K°). More comprehensive studies with DCCD and anoxia showed nearly complete inhibition of the active component of K⁺ influx over a wide range of K°, with no effect on the apparent permeability constant. DCCD had no effect on the electrogenic component of the cell potential (Ψ_p) above 0.2 millimolar K°. Net proton efflux was rapidly reduced 80 to 90% by DCCD. Since tissue ATP content and respiration were only slightly affected by the DCCD-pretreatment, the inhibitions of active K⁺ influx and Ψ_p at low K° can be attributed to inhibition of the plasmalemma ATPase.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Absence of a Formate-Induced Release of Bicarbonate from Photosystem II

Formate has been proposed to inhibit electron flow in photosystem II by replacing endogenous bound bicarbonate on the reaction center complex. A mass spectrometer was used to measure directly the CO₂/HCO₃⁻ released when maize thylakoids, showing normal rates of electron flow, were treated with formate. Although the formate inhibited electron flow by 95%, no release (displacement) of CO₂/HCO₃⁻ was detected. This is consistent with the concept that membrane-bound HCO₃⁻ is not a requirement for normal rates of electron flow through photosystem II. Moreover, formate and other monovalent anions do not inhibit electron flow by removing bound HCO₃⁻ but by binding to empty sites. The “bicarbonate effect” is a reversal, by high concentrations of exogenous bicarbonate, of anion inhibition of photosystem II.     

Methane and Trichloroethylene Degradation by Methylosinus trichosporium OB3b Expressing Particulate Methane Monooxygenase

Whole-cell assays of methane and trichloroethylene (TCE) consumption have been performed on Methylosinus trichosporium OB3b expressing particulate methane monooxygenase (pMMO). From these assays it is apparent that varying the growth concentration of copper causes a change in the kinetics of methane and TCE degradation. For M. trichosporium OB3b, increasing the copper growth concentration from 2.5 to 20 μM caused the maximal degradation rate of methane (V_max) to decrease from 300 to 82 nmol of methane/min/mg of protein. The methane concentration at half the maximal degradation rate (K_s) also decreased from 62 to 8.3 μM. The pseudo-first-order rate constant for methane, V_max/K_s, doubled from 4.9 × 10⁻³ to 9.9 × 10⁻³ liters/min/mg of protein, however, as the growth concentration of copper increased from 2.5 to 20 μM. TCE degradation by M. trichosporium OB3b was also examined with varying copper and formate concentrations. M. trichosporium OB3b grown with 2.5 μM copper was unable to degrade TCE in both the absence and presence of an exogenous source of reducing equivalents in the form of formate. Cells grown with 20 μM copper, however, were able to degrade TCE regardless of whether formate was provided. Without formate the V_max for TCE was 2.5 nmol/min/mg of protein, while providing formate increased the V_max to 4.1 nmol/min/mg of protein. The affinity for TCE also increased with increasing copper, as seen by a change in K_s from 36 to 7.9 μM. V_max/K_s for TCE degradation by pMMO also increased from 6.9 × 10⁻⁵ to 5.2 × 10⁻⁴ liters/min/mg of protein with the addition of formate. From these whole-cell studies it is apparent that the amount of copper available is critical in determining the oxidation of substrates in methanotrophs that are expressing only pMMO.     

Characterization of the na-requirement in cyanobacterial photosynthesis

The Na⁺ requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li⁺ concentration was examined in air-grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O₂ evolution) as a function of Na⁺ concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na⁺ (K_½[Na⁺]) were calculated. The value of both the low and the high K_½(Na⁺) was dependent upon extracellular DIC concentration. The low K_½(Na⁺) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K_½(Na⁺) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na⁺ and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na⁺ and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO₂ and HCO₃⁻ transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO₂ and HCO₃⁻ transport systems required Na⁺, directly or indirectly, for full activity. However, micromolar levels of Na⁺ were required for CO₂ transport while millimolar levels were required for HCO₃⁻ transport. These levels corresponded to those found for the low and high K_½(Na⁺) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO₂ transport increased with increased DIC concentration relative to HCO₃⁻ transport. This change was reflected in a decrease in the Na⁺ concentration required for maximum photosynthesis, in accord with the lower Na⁺-requirement for CO₂ transport. Lithium competitively inhibited Na⁺-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na⁺-dependent HCO₃⁻ transport. Lithium had little effect on CO₂ transport and only a small effect on the size of the pool it generated. Thus, CO₂ transport did not require a functional HCO₃⁻ transport system for full activity. Based on these observations and the differential requirement for Na⁺ in the CO₂ and HCO₃⁻ transport system, it was proposed that CO₂ and HCO₃⁻ were transported across the membrane by different transport systems.     

Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes

Effect of several parameters on inhibition of potato (Solanum tuberosum) invertase by its endogenous proteinaceous inhibitor was determined using homogeneous preparations of both proteins. The inhibitor and invertase formed an inactive complex with an observed association rate constant at pH 4.70 and 37°C of 8.82 × 10² per molar per second and a dissociation rate constant of 3.3 × 10⁻³ per minute. The inhibitor appeared to bind to invertase in more than one step. Initial interaction (measured by loss of invertase activity) was rapid, relatively weak, readily reversible (K_i of 2 × 10⁻⁶ molar) and noncompetitive with substrate at pH 4.70. Initial interaction was probably followed by isomerization to a tighter (K_i of 6.23 × 10⁻⁸ molar) complex, which dissociated slowly with a half-time of 3.5 hour. Interaction between enzyme and inhibitor appeared to be of ionic character and essentially pH independent between pH 3.5 and 7.4.     

Enzymic Properties of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Purified from Rice Leaves

The enzymic properties of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase purified from rice (Oryza sativa L.) leaves were studied. Rice RuBPcarboxylase, activated by preincubation with CO₂ and Mg²⁺ like other higher plant carboxylases, had an activation equilibrium constant (K_cK_Mg) of 1.90 × 10⁵ to 2.41 × 10⁵ micromolar² (pH 8.2 and 25°C). Kinetic parameters of carboxylation and oxygenation catalyzed by the completely activated enzyme were examined at 25°C and the respective optimal pHs. The K_m(CO₂), K_m(RuBP), and V_max values for carboxylation were 8 micromolar, 31 micromolar, and 1.79 units milligram⁻¹, respectively. The K_m(O₂), K_m(RuBP), and V_max values for oxygenation were 370 micromolar, 29 micromolar, and 0.60 units milligram⁻¹, respectively.

Comparison of rice leaf RuBP carboxylase with other C₃ plant carboxylases showed that it had a relatively high affinity for CO₂ but the lowest catalytic turnover number (V_max) among the species examined.

     

Activation state of ribulose bisphosphate carboxylase in soybean leaves

Conditions for extraction and assay of ribulose-1,5-bisphophate carboxylase present in an in vivo active form (initial activity) and an inactive form able to be activated by Mg²⁺ and CO₂ (total activity) were examined in leaves of soybean, Glycine max (L.) Merr. cv Will. Total activity was highest after extracts had preincubated in NaHCO₃ (5 millimolar saturating) and Mg²⁺ (5 millimolar optimal) for 5 minutes at 25°C or 30 minutes at 0°C before assay. Initial activity was about 70% of total activity. K_act (Mg²⁺) and K_act (CO₂) were approximately 0.3 millimolar and 36 micromolar, respectively. The carry-over of endogenous Mg²⁺ in the leaf extract was sufficient to support considerable catalytic activity. While Mg²⁺ was essential for both activation and catalysis, Mg²⁺ levels greater than 5 millimolar were increasingly inhibitory of catalysis. Similar inhibition by high Mg²⁺ was also observed in filtered, centrifuged, or desalted extracts and partially purified enzyme. Activities did not change upon storage of leaves for up to 4 hours in ice water or liquid nitrogen before homogenization, but were about 20% higher in the latter. Activities were also stable for up to 2 hours in leaf extracts stored at 0°C. Initial activity quickly deactivated at 25°C in the absence of high CO₂. Total activity slowly declined irreversibly upon storage of leaf homogenate at 25°C.     

Chemolithotrophic acetogenic H2/CO2 utilization in Italian rice field soil

Acetate oxidation in Italian rice field at 50 °C is achieved by uncultured syntrophic acetate oxidizers. As these bacteria are closely related to acetogens, they may potentially also be able to synthesize acetate chemolithoautotrophically. Labeling studies using exogenous H₂ (80%) and ¹³CO₂ (20%), indeed demonstrated production of acetate as almost exclusive primary product not only at 50 °C but also at 15 °C. Small amounts of formate, propionate and butyrate were also produced from ¹³CO₂. At 50 °C, acetate was first produced but later on consumed with formation of CH₄. Acetate was also produced in the absence of exogenous H₂ albeit to lower concentrations. The acetogenic bacteria and methanogenic archaea were targeted by stable isotope probing of ribosomal RNA (rRNA). Using quantitative PCR, ¹³C-labeled bacterial rRNA was detected after 20 days of incubation with ¹³CO₂. In the heavy fractions at 15 °C, terminal restriction fragment length polymorphism, cloning and sequencing of 16S rRNA showed that Clostridium cluster I and uncultured Peptococcaceae assimilated ¹³CO₂ in the presence and absence of exogenous H₂, respectively. A similar experiment showed that Thermoanaerobacteriaceae and Acidobacteriaceae were dominant in the ¹³C treatment at 50 °C. Assimilation of ¹³CO₂ into archaeal rRNA was detected at 15 °C and 50 °C, mostly into Methanocellales, Methanobacteriales and rice cluster III. Acetoclastic methanogenic archaea were not detected. The above results showed the potential for acetogenesis in the presence and absence of exogenous H₂ at both 15 °C and 50 °C. However, syntrophic acetate oxidizers seemed to be only active at 50 °C, while other bacterial groups were active at 15 °C.     

Na-Stimulation of Photosynthesis in the Cyanobacterium Synechococcus UTEX 625 Grown on High Levels of Inorganic Carbon

Photosynthesis of washed cells of Synechococcus UTEX 625 grown on 5% CO₂ was markedly stimulated (647 ± 50%) at pH 8.0 by the addition of low concentrations of NaCl (concentration required for half-maximal response, K_½, = 18 micromolar). Studies with KCl and Na₂SO₄ showed that the stimulation was due to Na⁺. Photosynthesis at pH 6.1 was only slightly stimulated by Na⁺. The response of photosynthesis at pH 8.0 to [Na⁺] was strongly sigmoidal for dissolved inorganic carbon ([DIC] ≤ 500 micromolar). Cells grown with high total [DIC], but air-levels of CO₂, at pH 9.6 showed the same response to low [Na⁺]. The absence of Na⁺ could be partially, but not completely overcome, by higher [DIC]. Various methods for examining CO₂ or HCO₃⁻ use (K_½^CO₂ determination; isotopic disequilibrium; and consideration of HCO₃⁻ dehydration rate) were consistent with CO₂ use by the cells, but HCO₃⁻ use could not be ruled out. Isotopic disequilibrium studies showed that CO₂ use was stimulated by Na⁺. Cells grown on 5% CO₂ accumulated DIC against a concentration gradient by a process (or processes) dependent on Na⁺. No evidence for uptake of Na⁺ concomitant with DIC uptake could be found. The lack of O₂ evolution during the initial and most rapid period of DIC accumulation suggested that the required energy was obtained from cyclic photophosphorylation.     

Effects of phosphorus deficiency on the photosynthesis and respiration of leaves of sugar beet

Phosphorus deficiency was induced in sugar beet plants (Beta vulgaris L. var. F5855441), cultured hydroponically under standardized environmental conditions, by removal of phosphorus from the nutrient supply at the ten leaf stage 28 days after germination. CO₂ and water vapor exchange rates of individual attached leaves were determined at intervals after P cutoff. Leaves grown with an adequate nutrient supply attained net rates of photosynthetic CO₂ fixation of 125 ng CO₂ cm⁻² sec⁻¹ at saturating irradiance, 25 C, and an ambient CO₂ concentration of about 250 μl l⁻¹. After P cutoff, leaf phosphorus concentrations decreased as did net rates of photosynthetic CO₂ uptake, photorespiratory evolution of CO₂ into CO₂-free air, and dark respiration, so that 30 days after cutoff these rates were about one-third of the control rates. The decrease in photosynthetic rates during the first 15 days after cutoff was associated with increased mesophyll resistance (r_m) which increased from 2.4 to 4.9 sec cm⁻¹, while from 15 to 30 days there was an increase in leaf (mainly stomatal) diffusion resistance (r_l′) from 0.3 to 0.9 sec cm⁻¹, as well as further increases in r_m to 8.5 sec cm⁻¹. Leaf diffusion resistance (r_l′) was increased greatly by low P at low but not at high irradiance, r_l′ for plants at low P reaching values as high as 9 sec cm⁻¹.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.     

An Efficient Preparation of Mulberroside A from the Branch Bark of Mulberry and Its Effect on the Inhibition of Tyrosinase Activity

A bioactive ingredient in an ethanol extract from the branch bark of cultivated mulberry Husang-32 (Morus multicaulis Perr.) was isolated using a macroporous resin column. The primary component, which was purified by semi-preparative high-performance liquid chromatography diode array detection (HPLC-DAD), was identified as mulberroside A (MA) by liquid chromatograph-mass spectrometer (LC-MS), ¹H and ¹³C nuclear magnetic resonance (NMR) spectra. In total, 4.12 g MA was efficiently extracted from one kilogram of mulberry bark. The enzymatic analysis showed that MA inhibited the generation of dopachrome by affecting the activities of monophenolase and diphenolase of tyrosinase in vitro. This analysis indicated that MA and oxyresveratrol (OR), which is the the aglycone of mulberroside A, exhibited strong inhibition of the monophenolase activity with IC₅₀ values of 1.29 µmol/L and 0.12 µmol/L, respectively. However, the former showed weaker inhibitory activity than the latter for diphenolase. For the monophenolase activity, the inhibitory activity of MA and OR was reversible and showed mixed type 1 inhibition. Additionally, the inhibition constant KI (the inhibition constant of the effectors on tyrosinase) values were 0.385 µmol/L and 0.926 µmol/L, respectively, and the KIS (the inhibition constants of the enzyme-substrate complex) values were 0.177 µmol/L and 0.662 µmol/L, respectively. However, MA showed competitive inhibition of diphenolase activity, and K_I was 4.36 µmol/L. In contrast, OR showed noncompetitive inhibition and K_I = K_IS = 2.95 µmol/L. Taken together, these results provide important information concerning the inhibitory mechanism of MA on melanin synthesis, which is widely used in whitening cosmetics.     

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella

Neither Dunaliella cells grown with 5% CO₂ nor their isolated chloroplasts had a CO₂ concentrating mechanism. These cells primarily utilized CO₂ from the medium because the K_(0.5) (HCO₃⁻) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K_(0.5) CO₂ was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO₂ concentrating mechanism was present that decreased the K_0.5 (CO₂) to about 0.5 micromolar and K_0.5 (HCO₃⁻) to 11 micromolar at pH 8. These K_0.5 values suggest that air-adapted cells preferentially concentrated CO₂ but could also use HCO₃⁻ from the medium. Chloroplasts isolated from air-adapted cells had a K_(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO₂. Chloroplasts from air-adapted cells, but not CO₂-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO₂ concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO₂ concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO₂ concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.     

Photosynthetic Activity of Chloroplasts Isolated From Bermudagrass (Cynodon dactylon L.), a Species With a High Photosynthetic Capacity

Chloroplasts have been isolated from bermudagrass (Cynodon dactylon L.) leaves and assayed for photophosphorylation and electron transport activity. These chloroplasts actively synthesize adenosine triphosphate during cyclic electron flow with phenazine methosulfate and noncyclic electron flow concurrent with the reduction of such Hill oxidants as nicotinamide adenosine dinucleotide phosphate, cytochrome c, and ferricyanide. Apparent Km values for the cofactors of photophosphorylation have been determined to be 5 × 10⁻⁵ M for phosphate and 2.5 × 10⁻⁵ M for adenosine diphosphate. The influence of light intensity on photophosphorylation has been studied and the molar ratio of cyclic to noncyclic phosphorylation calculated. It is concluded that the high photosynthetic capacity of bermudagrass leaves probably could be supported by the photophosphorylation capacities indicated in these chloroplast studies and the anomalous lack of data in chlorolast studies on the production of sufficient reductant for CO₂ assimilation at high light intensities has been noted.     

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K_m and V_max values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 μM. The K_m and V_max values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 μM. The apparent K_m for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K_m for H₂ uptake by cultures of Methanobacterium formicicum was 6 μM dissolved H₂. Formate and H₂ were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H₂ uptake was severely depressed when formate was above saturation and the dissolved H₂ was below 6 μM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.     

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : I. FACTORS AFFECTING NET CO(2) UPTAKE IN INTACT LEAVES AND CONTRIBUTION FROM RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE MEASURED IN VIVO AND IN VITRO

As part of an extensive analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C₃ grass, we have examined the response of leaf gas exchange and ribulose-1,5-bisphosphate (RuBP) carboxylase activity to temperature. Emphasis was placed on elucidating the specific processes which regulate the temperature response pattern. The inhibitory effects of above-optimal temperatures on net CO₂ uptake were fully reversible up to 40°C. Below 40°C, temperature inhibition was primarily due to O₂ inhibition of photosynthesis, which reached a maximum of 65% at 45°C. The response of stomatal conductance to temperature did not appear to have a significant role in determining the overall temperature response of photosynthesis. The intracellular conductance to CO₂ increased over the entire experimental temperature range, having a Q₁₀ of 1.2 to 1.4. Increases in the apparent Michaelis constant (K_c) for RuBP carboxylase were observed in both in vitro and in vivo assays. The Q₁₀ values for the maximum velocity (V_max) of CO₂ fixation by RuBP carboxylase in vivo was lower (1.3-1.6) than those calculated from in vitro assays (1.8-2.2). The results suggest that temperature-dependent changes in enzyme capacity may have a role in above-optimum temperature limitations below 40°C. At leaf temperatures above 40°C, decreases in photosynthetic capacity were partially dependent on temperature-induced irreversible reductions in the quantum yield for CO₂ uptake.     

Photosynthetic Adaptation by Synechococcus leopoliensis in Response to Exogenous Dissolved Inorganic Carbon

Synechococcus leopoliensis was grown over a wide range of dissolved inorganic carbon (DIC) concentrations (4-25,000 micromolar) which were obtained by varying culture pH (6.2-9.6) and the CO₂ concentration of the gas stream (36-50,000 microliters per liter). The [DIC] required to half-saturate photosynthesis (K_½^DIC) was found to vary depending upon the ambient DIC concentration at which the cells were grown. Low [DIC] grown cells exhibited low values of K_½^DIC (4.7 micromolar) whereas cells grown at high [DIC] exhibited high values of K_½^DIC (1-2.5 millimolar). Intermediate concentrations of DIC produced intermediate values. Changes in K_½^DIC appeared to be solely a function of [DIC] and were independent of both culture pH and CO₂ concentration. As changes in K_½^DIC occur in response to DIC concentrations commonly found in natural systems we suggest this adaptation may be of ecological significance.     

Modeling of the pyruvate production with<Emphasis Type="Italic"> Escherichia coli</Emphasis> in a fed-batch bioreactor

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q_VG=10 mL h^–1. Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q_VG=20 and 30 mL h^–1, respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking–Piret/Levenspiel term).List of symbols c_A acetate concentration (g L^–1) - c_A,0 acetate concentration in the feed (g L^–1) - c_G glucose concentration (g L^–1) - c_G,0 glucose concentration in the feed (g L^–1) - c_P pyruvate concentration (g L^–1) - c_P,max critical pyruvate concentration above which reaction cannot proceed (g L^–1) - c_X biomass concentration (g L^–1) - K_I inhibition constant for pyruvate production (g L^–1) - K_I^A inhibition constant for biomass growth on acetate (g L^–1) - K_P saturation constant for pyruvate production (g L^–1) - K_P inhibition constant of Jerusalimsky (g L^–1) - K_S^A Monod growth constant for acetate (g L^–1) - K_S^G Monod growth constant for glucose (g L^–1) - m_A maintenance coefficient for growth on acetate (g g^–1 h^–1) - m_G maintenance coefficient for growth on glucose (g g^–1 h^–1) - n constant of extended Monod kinetics (Levenspiel) (–) - q_V volumetric flow rate (L h^–1) - q_VA volumetric flow rate of acetate (L h^–1) - q_VG volumetric flow rate of glucose (L h^–1) - r_A specific rate of acetate consumption (g g^–1 h^–1) - r_G specific rate of glucose consumption (g g^–1 h^–1) - r_P specific rate of pyruvate production (g g^–1 h^–1) - r_P,max maximum specific rate of pyruvate production (g g^–1 h^–1) - t time (h) - V reaction (broth) volume (L) - Y_P/G yield coefficient pyruvate from glucose (g g^–1) - Y_X/A yield coefficient biomass from acetate (g g^–1) - Y_X/A,max maximum yield coefficient biomass from acetate (g g^–1) - Y_X/G yield coefficient biomass from glucose (g g^–1) - Y_X/G,max maximum yield coefficient biomass from glucose (g g^–1) - growth associated product formation coefficient (g g^–1) - non-growth associated product formation coefficient (g g^–1 h^–1) - specific growth rate (h^–1) - _max maximum specific growth rate (h^–1)